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Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing.
Protein Eng. 1998 Mar; 11(3):233-41.PE

Abstract

An expression system for mono- and bivalent single-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli. The cDNA encoding the Fv fragment of the anti-D monoclonal antibody D10 was cloned using the polymerase chain reaction and expressed in E.coli by fusing with a peptide tag link in the C-terminus of the light chain variable region. The scFv fragment expressed by the bacteria produced specific agglutination of human D positive red cells in the presence of an anti-peptide tag antibody. Flow cytometric analysis clearly indicated that the bacterially prepared scFv showed high specificity and affinity for D antigen, which was identical with that of the parental IgG. In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, bacterial alkaline phosphatase (BAP). The fusion protein produced significant agglutination of human red blood cells with D antigen, confirming that the bacterially expressed fusion protein is a functional bivalent antibody fragment. Specific agglutination of D positive red cells by D10 scFv-BAP was enhanced in the presence of anti-BAP antibody, suggesting that further multimerization of scFv led to highly efficient cell agglutination. By grafting BAP enzymatic activity into the scFv fragment (enzyme-linked scFv), blood typing could conveniently be performed. These results indicate that bacterially expressed scFv and scFv-BAP would be of practical use in blood typing. The system reported here could also be applied to the examination of other cell surface antigens and cell agglutination.

Authors+Show Affiliations

Japanese Red Cross Central Blood Center, Hiroo, Tokyo.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9613848

Citation

Furuta, M, et al. "Construction of Mono- and Bivalent Human Single-chain Fv Fragments Against the D Antigen in the Rh Blood Group: Multimerization Effect On Cell Agglutination and Application to Blood Typing." Protein Engineering, vol. 11, no. 3, 1998, pp. 233-41.
Furuta M, Uchikawa M, Ueda Y, et al. Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing. Protein Eng. 1998;11(3):233-41.
Furuta, M., Uchikawa, M., Ueda, Y., Yabe, T., Taima, T., Tsumoto, K., Kojima, S., Juji, T., & Kumagai, I. (1998). Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing. Protein Engineering, 11(3), 233-41.
Furuta M, et al. Construction of Mono- and Bivalent Human Single-chain Fv Fragments Against the D Antigen in the Rh Blood Group: Multimerization Effect On Cell Agglutination and Application to Blood Typing. Protein Eng. 1998;11(3):233-41. PubMed PMID: 9613848.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing. AU - Furuta,M, AU - Uchikawa,M, AU - Ueda,Y, AU - Yabe,T, AU - Taima,T, AU - Tsumoto,K, AU - Kojima,S, AU - Juji,T, AU - Kumagai,I, PY - 1998/6/5/pubmed PY - 1998/6/5/medline PY - 1998/6/5/entrez SP - 233 EP - 41 JF - Protein engineering JO - Protein Eng. VL - 11 IS - 3 N2 - An expression system for mono- and bivalent single-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli. The cDNA encoding the Fv fragment of the anti-D monoclonal antibody D10 was cloned using the polymerase chain reaction and expressed in E.coli by fusing with a peptide tag link in the C-terminus of the light chain variable region. The scFv fragment expressed by the bacteria produced specific agglutination of human D positive red cells in the presence of an anti-peptide tag antibody. Flow cytometric analysis clearly indicated that the bacterially prepared scFv showed high specificity and affinity for D antigen, which was identical with that of the parental IgG. In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, bacterial alkaline phosphatase (BAP). The fusion protein produced significant agglutination of human red blood cells with D antigen, confirming that the bacterially expressed fusion protein is a functional bivalent antibody fragment. Specific agglutination of D positive red cells by D10 scFv-BAP was enhanced in the presence of anti-BAP antibody, suggesting that further multimerization of scFv led to highly efficient cell agglutination. By grafting BAP enzymatic activity into the scFv fragment (enzyme-linked scFv), blood typing could conveniently be performed. These results indicate that bacterially expressed scFv and scFv-BAP would be of practical use in blood typing. The system reported here could also be applied to the examination of other cell surface antigens and cell agglutination. SN - 0269-2139 UR - https://www.unboundmedicine.com/medline/citation/9613848/Construction_of_mono__and_bivalent_human_single_chain_Fv_fragments_against_the_D_antigen_in_the_Rh_blood_group:_multimerization_effect_on_cell_agglutination_and_application_to_blood_typing_ DB - PRIME DP - Unbound Medicine ER -